The purpose of this project is to isolate the genes encoding the non-muscle myosins of Acanthamoeba and to use the genes as tools to investigate myosin structure/function relationships and the in vivo functions of these cytoplasmic myosins. Acanthamoeba expresses simultaneously at least three distinct myosin enzymes, myosin IA, myosin IB and myosin II. Using molecular cloning techniques, we have isolated and purified a myosin II heavy chain gene and a myosin IB heavy chain gene. While non-muscle and muscle myosins share many common features, non-muscle myosins do possess unique structural, enzymatic, and regulatory properties. The amoeba myosin sequence data has been of great value in furthering our understanding of the unique structural and functional aspects of the amoeba myosins. The significance of this work is that by using the tools of molecular biology we can approach the study of these myosins in novel ways which are not possible using the classical techniques of protein chemistry. For example, we can study protein structure/function relationships via site-directed mutagenesis of the gene and we can explore the in vivo functions of these myosins by reintroducing the genes back into amoeba.